This project proposes to establish a pipeline for the preclinical development of highly active and safe microbicides to prevent rectal transmission of HIV infection. For this project, the term 'preclinical' includes drug-development efforts progressing from in vitro testing of pre-selected candidate rectal microbicides in cell lines, followed by use of single cell suspensions of peripheral blood mononuclear cells and mucosal mononuclear cells, and ending with the use of intestinal explants. Aim 1 of this project is to define potential mucosal target cells for HIV infection and to characterize dissemination of migratory cells, with the potential to carry HIV, from rectal mucosa to other lymphoid tissue reservoirs. These data will help define the requisite profile of a successful rectal microbicide. Intestinal explant cultures obtained from surgical and biopsy specimens will be used to define the population of susceptible target cells within the rectal mucosa and the phenotype of cells emigrating from rectal tissue with the capacity to disseminate HIV to draining lymph nodes. Studies will include a broad range of viruses including R5 and X4 utilizing HIV and non clade B strains to determine the clade specificity of mucosal pathogenesis allied to the requirements of microbicide development. Aim 2 will be to evaluate the efficacy of potential rectal microbicide candidates, alone and in combination against HIV infection using cell-based assays and explant cultures. Initial studies will focus on the three antiretroviral reverse transcriptase inhibitor (RT) compounds: PMPA, UC-781 and TMC-120 alone and when formulated as gels. Aim 3 will be to evaluate the tissue biocompatibility of these rectal microbicide candidates, alone, and in combination, using cell based assays and intestinal explant cultures. The biocompatibility of individual compounds and combinations, with and without formulation, will be assessed by measurement of potential to,city using a broad range of cellular, histopathological, immunological and molecular assays using primary cells and rectal explants. Aim 4 will be to evaluate the efficacy of candidate rectal microbicides against rectal challenge with SIV (in the case of PMPA) and infectious RT-SHIV (in the case of UC-781 and TMC-120) in the Rhesus macaque model of SIV/RT-SHIV infection. The RT microbicides will be evaluated alone and in combination in the macaque model. These Aims will distill and establish preclinical steps in rectal microbicide drug development which will then transition to Project by Corner in which exploratory safety evaluation of the same compounds will occur in human studies.